Analytical validation of a 12-gene molecular test for the prediction of distant recurrence in breast cancer.

Aim: To validate the analytical performance of a 12-gene molecular assay that predicts distant recurrence for early-stage ER+/HER2- invasive breast cancer as run within a central reference laboratory.

Materials & Methods: Formalin-fixed paraffin-embedded breast resections were evaluated by quantitative reverse transcription polymerase chain reaction for the expression of eight target genes, three housekeeper genes and one control gene to assess for DNA contamination.

Results: The assay results were highly correlated with a validated reference laboratory.

Diagnostic Distinction of Malignant Melanoma and Benign Nevi by a Gene Expression Signature and Correlation to Clinical Outcomes.

Histopathologic examination alone can be inadequate for diagnosis of certain melanocytic neoplasms. Recently, a 23-gene expression signature was clinically validated as an ancillary diagnostic test to differentiate benign nevi from melanoma. The current study assessed the performance of this test in an independent cohort of melanocytic lesions against clinically proven outcomes.

An independent validation of a gene expression signature to differentiate malignant melanoma from benign melanocytic nevi.

Background: Recently, a 23-gene signature was developed to produce a melanoma diagnostic score capable of differentiating malignant and benign melanocytic lesions. The primary objective of this study was to independently assess the ability of the gene signature to differentiate melanoma from benign nevi in clinically relevant lesions.

Methods: A set of 1400 melanocytic lesions was selected from samples prospectively submitted for gene expression testing at a clinical laboratory.

Analytical validation of a proliferation-based molecular signature used as a prognostic marker in early stage lung adenocarcinoma.

Aims: The aim of these studies was to validate the analytical performance of a cell cycle progression (CCP) gene signature that provides prognostic information for early stage lung adenocarcinomas.

Materials & Methods: Formalin-fixed paraffin-embedded (FFPE) lung resections were evaluated by quantitative RT-PCR for the expression of 31 target and 15 housekeeper genes comprising the CCP score.

Results: The signature had a standard deviation (SD) of 0.

Analytical validation of a melanoma diagnostic gene signature using formalin-fixed paraffin-embedded melanocytic lesions.

Aim: These studies were to validate the analytical performance of a gene expression signature that differentiates melanoma and nevi, using RNA expression from 14 signature genes and nine normalization genes that generates a melanoma diagnostic score (MDS).

Materials & Methods: Formalin-fixed paraffin-embedded melanocytic lesions were evaluated in these studies.

Results: The overall SD of the assay was determined to be 0.

Clinical validation of a gene expression signature that differentiates benign nevi from malignant melanoma.

Background: Histopathologic examination is sometimes inadequate for accurate and reproducible diagnosis of certain melanocytic neoplasms. As a result, more sophisticated and objective methods have been sought. The goal of this study was to identify a gene expression signature that reliably differentiated benign and malignant melanocytic lesions and evaluate its potential clinical applicability.

Effects of ADARs on small RNA processing pathways in C. elegans.

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wild-type and ADAR mutant Caenorhabditis elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels.

Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer.

MicroRNAs (miRNAs) are small regulatory RNAs that are essential in all studied metazoans. Research has focused on the prediction and identification of novel miRNAs, while little has been done to validate, annotate, and characterize identified miRNAs. Using Illumina sequencing, ∼20 million small RNA sequences were obtained from Caenorhabditis elegans.

Role of RNA structure in regulating pre-mRNA splicing.

Pre-mRNA splicing involves removing non-coding introns from RNA transcripts. It is carried out by the spliceosome, along with other auxiliary factors. In general, research in splicing has focused on the sequences within the pre-mRNA, without considering the structures that these sequences might form.

Pentamidine reverses the splicing defects associated with myotonic dystrophy.

Myotonic dystrophy (DM) is a genetic disorder caused by the expression (as RNA) of expanded CTG or CCTG repeats. The alternative splicing factor MBNL1 is sequestered to the expanded RNA repeats, resulting in missplicing of a subset of pre-mRNAs linked to symptoms found in DM patients. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated.

Effect of a domain-spanning disulfide on aminoacyl-tRNA synthetase activity.

Enzymes regulated by allostery undergo conformational rearrangement upon binding effector molecules. For modular proteins, a flexible interface may mediate reorientation of the protein domains and transmit binding events to activate catalysis at a distance. Aminoacyl-tRNA synthetases (aaRSs) that use tRNA anticodons as identity elements can be considered allosteric enzymes in which aminoacylation of the tRNA acceptor stem is enhanced upon anticodon binding.

The protein factors MBNL1 and U2AF65 bind alternative RNA structures to regulate splicing.

Myotonic dystrophy type 1 (DM1) is a genetic disorder linked to a (CTG)(n) repeat expansion in the 3' untranslated region of the DMPK gene. Upon transcription in the nucleus, the CUG repeats form a stable RNA stem-loop that sequesters the RNA-binding protein MBNL1 from its normal function in the cell. MBNL1 regulates the alternative splicing of many pre-mRNAs, and upon MBNL1's sequestration, the alternative splicing of many genes is mis-regulated, leading to disease symptoms.

Identification of motifs that function in the splicing of non-canonical introns.

Background: While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract.

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T.

Myotonic dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the RNA splicing factor muscleblind-like (MBNL) is sequestered to the expanded CUG or CCUG RNAs. Mislocalization of MBNL results in missplicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients.